© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1313-1323 doi:10.1242/dev.104646
RESEARCH ARTICLE
Ontogeny, conservation and functional significance of maternally
inherited DNA methylation at two classes of non-imprinted genes
ABSTRACT
A functional role for DNA methylation has been well-established at
imprinted loci, which inherit methylation uniparentally, most commonly
from the mother via the oocyte. Many CpG islands not associated
with imprinting also inherit methylation from the oocyte, although the
functional significance of this, and the common features of the genes
affected, are unclear. We identify two major subclasses of genes
associated with these gametic differentially methylated regions
(gDMRs), namely those important for brain and for testis function.
The gDMRs at these genes retain the methylation acquired in the
oocyte through preimplantation development, but become fully
methylated postimplantation by de novo methylation of the paternal
allele. Each gene class displays unique features, with the gDMR
located at the promoter of the testis genes but intragenically for the
brain genes. Significantly, demethylation using knockout, knockdown
or pharmacological approaches in mouse stem cells and fibroblasts
resulted in transcriptional derepression of the testis genes, indicating
that they may be affected by environmental exposures, in either
mother or offspring, that cause demethylation. Features of the brain
gene group suggest that they might represent a pool from which
many imprinted genes have evolved. The locations of the gDMRs, as
well as methylation levels and repression effects, were also
conserved in human cells.
KEY WORDS: Epigenetics, DNA methylation, Imprinting
INTRODUCTION
DNA methylation in mammals refers to the modification of cytosine
by the addition of a methyl group, usually at the CpG dinucleotide.
Because methylcytosine spontaneously deaminates at very high
frequency, methylated cytosines are lost from the germline genome
(Walsh and Xu, 2006; Weber et al., 2007), resulting in underrepresentation except at CpG islands (CGIs). These are normally
unmethylated and under strong selective pressure to remain so. CGIs
are sometimes classed as ‘weak’ or ‘strong’, depending on the CpG
density and are frequently associated with the transcriptional start
1
Centre for Molecular Biosciences, School of Biomedical Sciences, University of
Ulster, Coleraine BT52 1SA, UK. 2Department of Maternal-Fetal Biology, National
Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya,
Tokyo 157-8535, Japan.
*Present address: Journal of Cell Science, The Company of Biologists, 140
Cowley Road, Cambridge CB4 0DL, UK.
‡
These authors contributed equally to this work
§
Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted
use, distribution and reproduction in any medium provided that the original work is properly
attributed.
Received 8 October 2013; Accepted 2 January 2014
sites of genes (Deaton and Bird, 2011). Classically, CGIs have been
identified by bioinformatic means but, more recently, biochemical
assays based on binding of the CXXC domain protein CFP1 have
been used to identify CGIs conserved between mouse and human,
almost half of which were not detected bioinformatically. Many
CFP1-defined CGIs are located away from annotated promoters in
intra- or intergenic locations, and have been termed ‘orphan’ CGIs
(Illingworth et al., 2010).
Although genome-wide sequencing approaches have increased
our knowledge of when and where methylation occurs, relatively
few classes of genes have been demonstrated to be functionally
regulated by methylation. One group of developmentally important
genes demonstrably controlled by methylation consists of imprinted
genes (Bartolomei and Ferguson-Smith, 2011). These are associated
with gametic differentially methylated regions (gDMRs), which
show different levels of methylation in sperm and egg, driven by the
activity of the de novo enzyme DNA methyltransferase 3A
(DNMT3A) and the essential co-factor DNMT3L (Shirane et al.,
2013), but with a possible small contribution from DNMT3B in
sperm (Kaneda et al., 2004). Loss of differential methylation causes
changes in transcription at the imprinted genes. Methylation of
imprinted gDMRs largely occurs (19/22 gDMRs) in the oocyte
(Reik and Walter, 2001; Proudhon et al., 2012).
Methylation patterns differ globally between the gametes, not just
at imprinted regions, and many non-imprinted loci have recently
been shown to have gDMRs (Kobayashi et al., 2012; Smallwood et
al., 2011). Following fertilisation, almost total erasure occurs on the
paternally inherited chromosomes however, probably mediated by
TET3 (Gu et al., 2011). For maternal chromosomes too there is a
passive loss of methylation by dilution, until it reaches a minimum
at the blastocyst stage. Following implantation de novo methylation
occurs, primarily driven by DNMT3B (Borgel et al., 2010). DNMT3
family members are barely detectable in most adult tissues except
testis and thymus, although DNMT3A is also found in brain (Okano
et al., 1999; Wu et al., 2010; Xu et al., 1999). Thus, propagation of
methylation patterns is thought to be reliant on the ubiquitous
maintenance enzyme DNMT1 (Howell et al., 2001; Li et al., 1992),
although DNMT3A/B may also play a role in embryonic stem cells
(ESCs) (Chen et al., 2003).
Despite some recent studies looking at loci inheriting methylation
from the mother (Borgel et al., 2010; Smallwood et al., 2011;
Kobayashi et al., 2012), a number of questions remain unresolved.
In particular, we aimed to: (1) determine if there are any gene
classes that are over-represented in these ~1000 oocyte-specific
gDMRs; (2) determine the functional significance of methylation at
these loci by removing it and examining the transcriptional
response; (3) examine the conservation of any gDMR between
human and mouse; (4) determine the methyltransferases important
for establishment and maintenance of the gDMR; and (5) compare
the gDMR properties with those of imprinted loci.
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Charlotte E. Rutledge1,*,‡, Avinash Thakur1,‡, Karla M. O’Neill1, Rachelle E. Irwin1, Shun Sato2, Ken Hata2 and
Colum P. Walsh1,§
We show here that there are three distinct types of oocyte gDMR:
those associated with imprinted genes and then two transient gDMR
classes, one containing genes associated with testis function and the
other with brain function. The former appear to be marked at the
promoter by DNA methylation, helping to suppress transcription.
The brain-specific genes are instead methylated intragenically.
Postfertilisation, both testis and brain gDMRs become fully
methylated by de novo methylation of the paternal allele. Testisspecific genes require methylation for complete repression, and in
its absence they become upregulated by several orders of magnitude.
Unlike imprinted genes, neither of the non-imprinted gDMR classes
requires germline passage to re-establish methylation.
RESULTS
Identification of genes that resemble imprinted genes in
their methylation ontogeny
We reanalysed recently published (Kobayashi et al., 2012;
Smallwood et al., 2011) datasets looking for CGIs that: (1) were
completely methylated (>75%) in wild-type (WT) meiosis II (MII)
oocytes; (2) were significantly methylated in blastocysts; and (3) lost
>50% methylation in DNMT3L-deficient oocytes. The threshold for
significant methylation in blastocysts was set to 25%, based on the
fact that many imprinted CGIs show methylation at ~30% in the
genome-wide analyses. In the first of these studies, which used
reduced representation bisulfite sequencing (RRBS) of oocytes in
MII, 2146 /23,019 CGIs examined were >75% methylated in
oocyte: after removing intergenic CGIs and accounting for multiple
CGIs associated with a single gene, this gave 897 RefSeq-annotated
genes with at least one completely methylated CGI (Fig. 1A). None
of these was methylated in sperm. In blastocyst, only 529 genes had
CGIs with significant methylation (Fig. 1A). Interestingly, most
genes (421/529, 80%) that were methylated at this level in blastocyst
were also methylated in oocyte, and more heavily methylated in
oocyte than in sperm, indicating that they are oocyte gametic
differentially methylated regions (oocyte gDMRs). Although we
distinguish (Fig. 1A) between CGIs that appear to gain methylation
preimplantation (108/529) and those that retain methylation from the
oocyte (421/529), it is possible that some in the latter group are both
demethylated early on and then remethylated to a lower degree on
both alleles. In contrast to the oocyte CGIs, only 12 sperm gDMRs
showed retention of methylation in the blastocyst. Indicating the
importance of DNMT3L, 91% (382/421) of the genes methylated in
both oocyte and blastocyst lost >50% of their methylation in oocytes
from Dnmt3l−/− females (Fig. 1A).
To determine whether any gene class was over-represented in this
group (n=382) of DNMT3L-dependent oocyte gDMR genes, we used
the functional annotation clustering tool on the DAVID platform
(Huang et al., 2009). The top-scoring groups of genes identified as
being enriched are indicated in Fig. 1Bi. This identified genes
associated with the term ‘brain’, such as Crocc, Grin3b and Adcy6
(total n=199), as markedly over-represented (P=5.76×10−11),
accounting for 52% of the total group: indeed, 4/5 top classes were
central nervous system genes (Fig. 1Bi). DAVID analysis of the genes
methylated in blastocyst (n=529) gave very similar results. Analysis
of an independent dataset derived from whole bisulfitome DNA
amplification-sequencing (WBA-seq) on germinal vesicle (GV) stage
oocytes (Kobayashi et al., 2012) was a close match, with identity in
4/5 classes and similar ranking despite the different approaches, stage
of maturity and numbers of CGIs examined (Fig. 1Bii). By contrast,
a study by Weber and colleagues employing methylated DNA
immunoprecipitation (meDIP) (Borgel et al., 2010) found that the
class of genes most significantly associated (P=5.6×10−5) with
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preimplantation methylation was the testis-specific genes (Fig. 1Biii)
and that some of these were oocyte gDMRs. This is likely to be due
in part to differences in technique, since only promoter regions were
examined in the meDIP study, and many of the testis genes had nonCGI promoters and thus were under-represented in the other studies.
Examination of the WGA-seq analysis, however, also indicated that
many testis-specific genes were methylated in oocyte (Kobayashi et
al., 2012), despite not being highlighted by ontogeny analysis. Reinspection confirmed that testis-specific genes, such as Dpep3, Piwil1,
Fkbp6, Rhox13, Trim52, Spag1, Ggn1, Tbpl1, Spata16, Csnka2ip and
Tssk2, identified in these two latter studies, do fulfil our criteria for
DNMT3L-mediated programming. Notably, the testis-specific genes
showed no overlap with the group of germline genes that are
methylated postimplantation by DNMT3B, such as Dazl, Sycp3 and
Tex12, identified earlier (Borgel et al., 2010; Hackett et al., 2012;
Maatouk et al., 2006; Weber et al., 2007). The latter group is
completely unmethylated in oocyte and blastocyst, unlike the testis
gDMR genes, is independent of DNMT3L status but dependent on
DNMT3B and contains genes expressed in oocyte as well as testis
(e.g. the Sycp genes).
The top-scoring class from our DAVID analyses includes a few
known imprinted genes expressed in brain (e.g. Snrpn, Gnas and
Ncdn, which were excluded from this group in later analyses),
suggesting the presence of novel imprinted genes. Many new brainspecific imprinted genes were recently reported from genome-wide
analyses (Gregg et al., 2010a; Gregg et al., 2010b), although only a
fraction have been independently validated (DeVeale et al., 2012).
Likewise, some of the testis-specific genes show allele-specific
methylation preimplantation (Borgel et al., 2010), a feature of
imprinted genes. Fig. 1C shows little overlap, however, between the
genes identified in those studies and the brain genes identified in the
RRBS dataset. Of the novel brain-specific imprinted genes identified
by others, only three (Ccdc40, Cdh15 and Herc3; Fig. 1C) also have
a DNMT3L-dependent gDMR: significantly, these are among the
minority that have been experimentally validated (DeVeale et al.,
2012; Gregg et al., 2010a; Proudhon et al., 2012). Our analysis
therefore suggested that there might be three classes of DNMT3Lprogrammed gDMR: those associated with imprinted genes, genes
important for brain function and genes that are specific to the testis.
Fig. 1D tracks the methylation ontogeny of each of the three
classes, as well as the germline genes methylated postimplantation,
based on approximately equal numbers of genes representative of
each class for which data are available from more than one study. All
three gDMR groups show strong dependence on DNMT3L in oocyte,
lower methylation in sperm and retention of methylation in blastocyst
(Fig. 1D). By contrast, the postimplantation group shows no
methylation at any of these stages. The brain gDMR displays higher
methylation in sperm and greater variability overall than the other two
groups (Fig. 1D). The CGI structure and location also differ between
groups. The gDMRs of the testis genes are: (1) generally the only CGI
present; (2) classical CGIs; and (3) always located at the proximal
promoter position upstream of the first exon (e.g. Rhox13; Fig. 1E).
By contrast, the brain gene had: (1) multiple CGIs; (2) gDMRs that
were always intragenic CGIs, usually in an exon; (3) gDMRs that
were non-classical or orphan CGIs, as defined by CFP1 binding; and
(4) an unmethylated promoter CGI (e.g. Crocc CGI 4-1220; Fig. 1E).
Confirming that brain gDMRs are largely intragenic rather than
promoter associated, only 14% (21/151) of brain gDMRs common to
both the RRBS and WGA-seq studies were at annotated RefSeq
transcription start sites (TSSs) (Fig. 1F). Furthermore, only 18%
(23/130) of the remainder were enriched for hypophosphorylated
RNA polymerase II and H3K4me3, indicative of TSSs (Illingworth et
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Development (2014) doi:10.1242/dev.104646
al., 2010), in mouse ESCs: in brain tissues this number fell to 11%
(14/130). The imprinted regions are more diverse but, as previously
noted (Chotalia et al., 2009), the gDMR is generally one of multiple
CGIs at these loci and tends to be intragenic. These results identify
three distinct classes of gDMR programmed by DNMT3L in the
mouse oocyte.
Dnmt3l is required for methylation and repression of some
testis genes in the female germline
We set about validating these bioinformatic data by collecting oocytes
from 22 days postpartum (22 dpp) WT and Dnmt3l−/− mice. Using
combined bisulfite treatment and restriction analysis (COBRA), which
produces digested bands only if a region is methylated, we first
confirmed the known DNMT3L-dependent methylation of the gDMR
at Snrpn, as a control, using oocytes from WT and Dnmt3l−/− females
(Fig. 2A). H19 shows no methylation, confirming the lack of somatic
cell contamination (Fig. 2A). The testis gDMR at Dpep3 and Piwil1,
previously identified by meDIP (Borgel et al., 2010), also showed
near-complete methylation in WT oocytes, further confirmed by
clonal analysis (Fig. 2A,B). The weak CGI at Csnka2ip, however,
only showed partial methylation in oocyte (Fig. 2A,B). Sperm
methylation was seen only at the control locus H19 (Fig. 2A). The
gDMRs all showed total loss of methylation in oocytes from
Dnmt3l−/− females (Fig. 2A), as confirmed by bisulfite sequencing
(examples in Fig. 2B). Two further testis-specific genes, Rhox13 and
Fkbp6, identified by RRBS (Smallwood et al., 2011), were also highly
methylated in oocyte, whereas Dazl, which is methylated
postimplantation by DNMT3B (Weber et al., 2007), was not
(Fig. 2C). Methylation at Fkbp6 in sperm (Fig. 2C) is in contrast to
earlier reports, but was confirmed by clonal analysis (supplementary
material Fig. S1). Fkbp6 is transcribed in spermatogonial stem cells
(Fig. 2D) but is switched off and presumably methylated as cells enter
meiosis. By contrast, Ddx4, which belongs to the postimplantation
group, is transcribed in postmeiotic spermatocytes (white arrowhead,
Fig. 2D) and not methylated in sperm.
We used pyrosequencing, a more quantitative method, to extend
our studies to brain gDMRs, as these show smaller differences in
methylation between sperm and oocyte (Fig. 1D). This assay gave
similar results to previous COBRA and clonal analyses for imprinted
(Peg1) and testis-specific (Rhox13) loci and confirmed that
intragenic gDMRs at brain-specific genes such as Grin3b, Adcy6
and Crocc show significantly different methylation between oocytes
and sperm (Fig. 2E).
In order to determine if demethylation of any of these sequences
results in transcriptional activation, we examined mRNA levels in
ovary by reverse-transcription quantitative PCR (RT-qPCR). This
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Fig. 1. Two novel gene classes with gametic
differentially methylated regions (gDMRs).
(A) Numbers of genes (n) showing methylation
(Meth) in oocyte, blastocyst or both from
reanalysis of published genome-wide data
(Smallwood et al., 2011). DNMT3L-dependent:
genes with >50% loss of methylation in
DNMT3L-deficient oocytes. (B) Top-ranking
classes of genes by ontology analysis of data
from: (i) reduced representation bisulfite
sequencing (RRBS) in meiosis II (MII) oocytes
(as in A); (ii) whole bisulfitome amplified DNA
sequencing on germinal vesicle (GV) oocytes
[WBA-seq (Kobayashi et al., 2012)]; and (iii)
methylated DNA immunoprecipitation (meDIP)
on preimplantation embryos (Borgel et al., 2010).
P-value and false discovery rate (FDR) are
indicated. (C) Top-scoring class from B(i)
compared with novel imprinted genes (De Veale
et al., 2012) and those identified by meDIP
[B(iii)]. (D) Methylation ontogeny of
representative groups of genes from each gDMR
class; genes methylated postimplantation are
also shown as a control. 3L KO, oocytes from
Dnmt3l−/− females; Blast, blastocyst. Error bars
represent s.e.m. (E) Structure of prototypical
brain (Crocc) and testis (Rhox13) gDMR genes.
Black boxes: CpG islands (CGIs), with ID
number, identified by CFP1 binding (Illingworth
et al., 2010). Green boxes: CGI identified by
conventional means, with number of CpGs
indicated. Pyro, region covered by
pyrosequencing assay. (F) Proportions of brain
gDMRs at known (RefSeq) transcriptional start
sites (TSSs), those showing TSS-associated
chromatin marks in ESCs or brain cells, and
those with no evidence of being TSSs.
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Fig. 2. Validation of methylation at novel gDMRs in gametes and effects
of DNMT3L loss. (A) COBRA of methylation in wild-type (WT) and DNMT3Ldeficient (3L) mouse oocytes. un, uncut; u, unmethylated; asterisk, limit
digest band. (B) Clonal analysis, with methylation as a percentage of all sites
indicated. Arrowheads indicate sites also analysed by COBRA. (C) As for A.
Post-impl, genes known to be methylated only after implantation. (D) In situ
hybridisation of testis tissue. Arrow indicates spermatogonial stem cells;
arrowhead indicates spermatozoa. (E) Methylation of gDMRs as detected by
pyrosequencing in sperm and oocytes. (F) RT-qPCR showing transcript
levels in WT and 3L KO ovaries for the indicated genes. Error bars indicate
s.e.m. **P<0.01, ***P<0.001 by t-test.
not derepressed in this tissue (Fig. 2F), despite showing levels of
methylation in Dnmt3l−/− oocytes as low as those seen in primordial
germ cells, where they reach their minimum (supplementary
material Fig. S2). As the brain gDMRs are not at promoters, they
were not assessed. These results validate the bioinformatic analysis
above and suggest a role for methylation in maintaining suppression
at some non-imprinted loci in oocyte.
showed a small but significant upregulation of Dpep3 and Piwil1 in
DNMT3L-deficient ovaries (Fig. 2F), the latter supporting a
previous observation (Kobayashi et al., 2012). By contrast, IAP was
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When we looked at postimplantation tissues, the Snrpn gDMR, as
expected for an imprinted gene, retained ~50% methylation in adult
somatic tissues (Fig. 3A). By contrast, all of the testis gDMRs
showed complete or near-complete methylation (Fig. 3A), indicating
a gain of methylation on the paternal allele. Methylation levels were
comparable to those seen at Sycp3 (Fig. 3A), part of the group
methylated postimplantation by DNMT3B (Weber et al., 2007).
Pyrosequencing confirmed these results and extended them to brain
gDMRs, which also showed near-complete methylation in adult
tissues (Fig. 3B), indicating that the brain and testis gDMRs show
transient, rather than stable, differential methylation. The similar
levels of methylation in tissues derived from all three germ layers
indicate establishment early postimplantation.
Although such high methylation is inconsistent with imprinting,
it could still be important for transcriptional repression. NIH 3T3
fibroblast cells showed levels of DNA methylation comparable to
primary tissues for most of the genes of interest (Fig. 3A, lanes at
right); treatment with the methyltransferase inhibitor 5-aza-2′deoxycytidine (Aza) caused demethylation (Fig. 3A,C) and strong
induction (Fig. 3D) of Sycp3 and IAP as expected (Borgel et al.,
2010; Walsh et al., 1998). All testis gDMRs showed substantial
demethylation by COBRA (Fig. 3A) and pyroassay (Fig. 3C;
P<0.05 for all genes) and were strongly induced (Fig. 3D) from
almost undetectable levels in untreated fibroblasts. Extension of this
analysis to cover additional testis gDMRs by RT-qPCR showed
substantial derepression at some other loci (Rhox9, Trim52, Spata16;
Fig. 3E). There was a notable gradation of response dependent on
CpG density in the gDMR: out of three testis genes containing weak
CGIs (Tssk2, Csnka2ip, Dnmt3l), only one (Dnmt3l) showed
significant (P=0.04) derepression (Fig. 3D,E); this effect on Dnmt3l
is consistent with previous reports by ourselves and others (Hu et
al., 2008; O’Doherty et al., 2011). By contrast, many of the brain
gDMR genes were less heavily repressed in fibroblasts and showed
substantial baseline transcription by RT-PCR, using brain as a
positive control (Fig. 3D). Consistent with brain gDMRs not being
cryptic promoters, Aza treatment gave demethylation (all P<0.05)
but no significant derepression, except for a slight (P=0.036) effect
at Adcy6 (Fig. 3D,E). These results show a postimplantation gain of
methylation on the paternal allele at brain and testis gDMRs and an
Development
Brain and testis gDMRs become fully methylated
postimplantation, with methylation required for suppression
of testis genes
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Development (2014) doi:10.1242/dev.104646
Fig. 3. Methylation and repression at novel gDMRs in adult mouse tissues. (A) COBRA on the adult mouse tissues and cell lines indicated at the top.
Fibro, fibroblasts without (–) or with (+aza) 5-aza-2′-deoxycytidine. (B) Methylation analysis by pyroassay. (C) All analysed genes showed significant
demethylation (P<0.05, ANOVA) by pyroassay following Aza treatment. (D) RT-PCR for the indicated genes in treated fibroblasts. Testis is a positive control
(Pos ctrl), except for Adcy6 and Grin3b, where brain was used. IAP and Sycp3 are known to be repressed by methylation. Asterisked genes have low-density
CGIs. Hprt is a loading control. (E) RT-qPCR of samples from D. UT, untreated. Error bars indicate s.e.m.
Conservation of methylation and repression in a human cell
line
We extended our study to hTERT-1604 immortalised human
fibroblasts, which have previously been shown to retain normal
methylation at many loci (Loughery et al., 2011). Methylation at the
SNRPN locus is within the 40-60% range (Fig. 4A) normal for
imprinted gDMRs (Woodfine et al., 2011). The testis-specific genes
DPEP3, PIWIL1 and FKBP6 showed high levels of conservation
between mouse and human, retained their promoter CGIs and were
heavily methylated (Fig. 4A). SYCP3 and DAZL were also heavily
methylated as expected, and are shown as controls (Fig. 4A). Many
brain genes also showed conservation of the intragenic CGI that
constitutes the murine gDMR. Methylation was >75% for GRIN3B
and ADCY6, as seen in mouse, although EFNA was not heavily
methylated in this cell line (Fig. 4A).
Contrasting with robust signal in testis, little or no transcription
from the testis gDMR group (DPEP3, FKBP6, PIWIL1) or from the
genes methylated postimplantation (SYCP3, SYCP1, DAZL, TEX12)
could be detected in untreated hTERT-1604 fibroblasts, as expected
(Fig. 4B). Humans lack a Rhox13 homologue, but RHOXF1, which
is related to the ancestral gene in mice, also showed the same
expression pattern (Fig. 4B). On treatment with Aza, all of the testis
loci except FKBP6 showed significant demethylation (P<0.05; Aza,
Fig. 4C), although it is notable that the gDMR appeared to be more
resistant (loss <10%) than the postimplantation genes (loss >15%).
All genes except PIWIL1 were reliably upregulated (Fig. 4B) and
RT-qPCR confirmed major differences in transcript levels versus
control (P<0.05; Fig. 4D). Brain genes showed low-level
transcription in fibroblasts and failed to show substantial
upregulation with Aza (not shown), as found in mouse (Fig. 3D).
Aza affects all three methyltransferases as well as having some nonspecific effects, so we also carried out siRNA knockdown for
DNMT1 and collected RNA at day 4. Significant (P<0.05)
demethylation at all the testis and postimplantation loci examined
(siRNA, Fig. 4C) correlated with upregulation of FKBP6, SYCP3
and DAZL (Fig. 4D). These results confirm that methylation plays
an evolutionarily conserved role in regulating testis genes identified
as gDMR in mice, as well as germline genes methylated
postimplantation.
Contribution of DNMT1 versus DNMT3A/B to maintenance
methylation of gDMRs
We used knockout (KO) ESC lines to assess the dependence of nonimprinted gDMRs on the maintenance versus de novo enzymes for
perpetuation of methylation. Parental J1 ESCs retain appropriate
methylation at the Snrpn, H19 and Peg1 gDMRs by COBRA
(Fig. 5A) and/or pyrosequencing (Fig. 5D), and clonal analysis
showed that Snrpn predominantly displays the two patterns typical
of imprinted genes, either fully methylated or fully unmethylated
(Fig. 5B). J1 ESCs lacking a functional Dnmt1 gene also showed
methylation loss at the imprinted loci, as expected (1KO,
Fig. 5A,D). Whereas all of the testis gDMRs showed levels of
methylation comparable to (Piwil1, Rhox13, Fkbp6) or greater than
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important role for methylation in maintaining suppression of the
latter.
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Fig. 4. Conservation of methylation and repression in human. (A) Methylation levels as determined by pyrosequencing in hTERT-1604 normal human
fibroblasts. (B) RT-PCR showing derepression by Aza treatment (lane +) is comparable between testis gDMR genes (left) and genes methylated
postimplantation (right). Adult testis is a positive control; ACTB is for loading. (C) Significant differences in methylation were seen at all analysed genes in cells
exposed to Aza or depleted of DNMT1 by siRNA (P<0.05, except for FKBP6 with Aza which was not significant). (D) RT-qPCR for the same samples. All
upregulation >5-fold was significant at P<0.05. Error bars indicate s.e.m.
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(P=0.05), respectively] in the 1KO cells (Fig. 5E). The other genes,
including Fkbp6, which showed the largest change (15-fold) in the
previous study, showed smaller but reproducible (4-fold in 3ab DKO
cells, P=0.027) alterations in transcript levels (Fig. 5E). There was
no significant derepression at brain gDMR genes. These results
suggest that DNMT1 contributes the most to repression at these loci
and that strategies aimed at inhibiting all three enzymes (such as
Aza treatment) should have the greatest effect on derepression.
Non-imprinted gDMRs do not require germline passage to
acquire de novo methylation
A number of studies have shown that imprinted gDMRs, which
acquire their methylation in the growing oocyte in a Dnmt3ldependent process, are unable to re-establish this methylation
outside of the female germline, once lost (Chen et al., 2003; Holm
et al., 2005; Tucker et al., 1996; Wernig et al., 2007). Since the brain
and testis gDMRs also acquire their methylation in the oocyte we
examined whether they too needed to pass through the germline for
successful reprogramming. We examined methylation in 1KO ESCs
rescued with a cDNA expressing full-length DNMT1 protein
(1KO+1) (Oda et al., 2006). As expected, rescued cells fail to
remethylate imprinted gDMRs (Fig. 6A,B; 1KO versus IKO+1,
P=0.57; not significant for Peg1); by contrast, all testis and brain
Development
(Dpep3, Csnka2ip) in blastocyst, they showed clear loss of
methylation in DNMT1 KO cells by COBRA (Fig. 5A) and
significant demethylation by pyroassay; Rhox13 is shown as an
example (Fig. 5D; WT versus 1KO, P=0.002). The same was true
for brain gDMRs, as assessed by pyroassay (Fig. 5D; all P<0.05).
Interestingly, although Fkbp6, like Snrpn, shows ~50% methylation
overall there is less clear partitioning of methylation into fully
methylated versus fully unmethylated alleles (Fig. 5B). The same
was true for the Grin3b brain gDMR (Fig. 5B). We also examined
methylation levels in two independent Dnmt3a−/− Dnmt3b−/− double
KO (DKO) lines, 16aabb and 7aabb (Okano et al., 1999). These
showed significant (P<0.05) loss of methylation at all testis and
brain gDMRs except Crocc, as well as at the imprinted gDMRs
(Fig. 5C,D). Our results confirm that, in the absence of these
enzymes, methylation cannot be maintained by DNMT1 alone.
Triple KO (TKO) ESCs lacking all three DNMT enzymes have
been previously reported to have 4- to 15-fold increases in Dpep3,
Fkbp6, Rhox13 and Dazl transcript levels and ~5-fold increases in
IAP levels (Karimi et al., 2011b). We used RT-qPCR in WT, 1KO
and 3ab DKO cells to separate the cell response to the loss of either
the maintenance enzyme or the de novo enzyme systems. The
greatest changes were seen at the Dpep3 and IAP loci in both cell
types, with greater derepression [10-fold (P=0.002) and 5-fold
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.104646
gDMRs, except Adcy6, showed significant gains in methylation
(Fig. 6A,B; 1KO versus 1KO+1, P<0.05).
As methylation was not restored to WT levels, we quantitated the
global increase in methylation in rescued cells using the LUMA
assay (Karimi et al., 2011a), which is a bisulfite-independent
quantitative assay that uses methylation-sensitive and -insensitive
restriction enzyme cleavage to estimate total genomic methylation
levels, and showed that 1KO+1 cells had an overall gain of 28%
versus 1KO cells (Fig. 6C). As the pyroassay appeared to be
showing slightly lower gains in methylation than COBRA, we also
carried out clonal analysis of representative genes (Fig. 6D;
supplementary material Fig. S3). Graphing these results (Fig. 6E)
confirms the difference between imprinted (no significant gain, P=1)
and non-imprinted (significant, P<0.05) loci, with the average
increase across the four non-imprinted gDMRs being 26%, in good
agreement with LUMA (Fig. 6C). In keeping with the re-
establishment of repression, RT-qPCR showed that transcript levels
were lower in 1KO+1 cells than in 1KO (not shown).
Levels of de novo methyltransferase activity in almost all adult
tissues are reported to be very low (Okano et al., 1998), implying
that methylation might not be restored at these non-imprinted
gDMRs in adult cells. To test this, we were able to significantly
deplete NIH 3T3 cells of DNMT1 using siRNA (Fig. 6F;
P=1.8×10−5, day 4 versus scrambled control), then allowed the
DNMT1 levels to recover and examined the response of the gDMR.
Four days after knockdown of DNMT1 there was a decrease in
methylation at all loci tested (P<0.05) due to loss of maintenance
activity (Fig. 6G; untreated levels set to 100%), with brain gDMRs
showing the greatest response. As DNMT1 levels recovered the
percentage of methylated sites increased, presumably due to its
maintenance activity stabilising the methylation added by the de
novo enzymes, as in ESCs [see above and Oda et al. (Oda et al.,
1319
Development
Fig. 5. Enzyme dependence for gDMR classes in mouse stem cells. (A) Methylation levels in parental J1 ESCs (WT) and DNMT1-deficient derivatives
(1KO) assessed by COBRA. un, uncut control. (B) Clonal analysis of a gDMR from an imprinted, testis or brain gene (Snrpn, Fkbp6 and Grin3b, respectively).
(C) COBRA showing methylation levels in clonally derived ESCs (16aabb, 7aabb) carrying double knockouts (DKO) in both Dnmt3a and Dnmt3b.
(D) Pyrosequencing for the indicated gDMRs. Blastocyst methylation levels are derived from the analysis shown in Fig. 1D. Differences between DKO and WT
were significant (P<0.05) for all analysed genes except Crocc. (E) Fold reactivation in mutant versus WT cells as assessed by RT-qPCR. 3ab, DKO cells (as
above); TKO, triple KO cells lacking DNMT1, DNMT3A and DNMT3B, data from Karimi et al. (Karimi et al., 2011b); brain and imprint gDMR unavailable. Error
bars indicate s.e.m.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.104646
2006)], until by 18 days there was no longer a significant difference
(all P>0.05) in methylation between untreated and treated cells
except at the Fkbp6 locus, which remained demethylated (P<0.007).
Examination of transcription levels showed that, following
derepression (P<0.05) of testis gDMR genes at day 4 (subsequently
set to 100%), the genes were silenced again rapidly (Fig. 6H).
DISCUSSION
Although a number of recent studies have identified transient
gDMRs methylated in the oocyte (Borgel et al., 2010; Kobayashi et
al., 2012; Proudhon et al., 2012; Smallwood et al., 2011; Smith et
al., 2012), the functional significance of the methylation and the
types of genes affected were not identified. We show here for the
first time that transient gDMRs fall into two classes, one containing
genes predominantly expressed in the testis and the other enriched
in genes associated with brain function. Both groups have higher
levels of methylation in the oocyte than in sperm, are very
dependent on DNMT3L and retain methylation at levels of 25% or
more in blastocyst (Fig. 7). Although they resemble imprinted genes
in these respects, they fail to maintain differential methylation in
differentiated somatic tissues due to de novo methylation of paternal
1320
alleles after implantation. As previously noted (Smallwood et al.,
2011; Proudhon et al., 2012), this highlights protection of the
unmethylated imprinted allele in the post-gastrulation embryo as one
of the most important properties of imprinted loci.
The difference between a transient gDMR described here and an
imprinted gDMR at a gene such as Cdh15 is not substantial, as the
latter gains methylation postimplantation on the paternal allele as
well as becoming biallelically expressed in certain tissues (Proudhon
et al., 2012). It has been previously noted that many imprinted genes
are expressed in brain (Wilkinson et al., 2007) and have intragenic
gDMRs (Smallwood and Kelsey, 2012). Since slightly more than
half of all transient gDMRs are associated with brain ontologies
(Fig. 1B) and are at CGIs old enough to show conservation between
human and mouse (Fig. 4), it seems reasonable to speculate that
some brain-specific imprinted genes might have arisen from this
particular group by, for example, alterations at a transient gDMR
locus that allow for protection of the paternal allele from de novo
methylation postimplantation.
With respect to the functional significance of methylation at the
transient gDMR, we show that demethylation causes transcriptional
derepression of testis genes in the Dnmt3l−/− ovary, Dnmt1−/− or
Development
Fig. 6. Reprogramming ability of the different gDMR classes. (A) Methylation by COBRA in ESCs with normal (WT), absent (1KO) and rescued (1KO+1)
DNMT1 status. (B) Pyroassay indicates significant (P<0.05) gain in methylation in 1KO+1 cells for all analysed genes except Peg1 and Adcy6. (C) Overall gain
in methylation in 1KO+1 cells assayed by LUMA. (D) Clonal analysis for one gene from each class of gDMR. (E) Analysis of clones indicates significant
(P<0.05) gain in methylation in 1KO+1 cells for all non-imprinted genes analysed. (F) Timecourse of Dnmt1 knockdown and recovery by RT-qPCR following
transient transfection of adult mouse fibroblasts with siRNA. Scr, scrambled control; d, days. (G) Pyroassay showing methylation loss and re-establishment for
samples shown in F; untreated set to 100%. All genes were significantly demethylated (P<0.05) at 4 days, but were no longer significantly different from
untreated (UT) at 18 days, except for Fkbp6. (H) RT-qPCR of same samples showing re-establishment of repression. Day 4 values were set to 100%. KD,
knockdown. Error bars indicate s.e.m.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.104646
Dnmt3ab−/− stem cells, and Aza-treated or DNMT1 siRNA-treated
adult cells, with the effect being much greater in the differentiated
somatic cells. It is possible that methylation of the maternal allele is
used to effectively halve the expression level of the transient gDMR
preimplantation: however, this effect is likely to be small at this
developmental stage, as derepression in KO ESCs is orders of
magnitude lower than in differentiated cells. Testis genes that are
regulated by a gDMR can be identified by the following
characteristics: they have a high density or ‘strong’ CGI at the
promoter, they are >75% methylated in oocytes, are DNMT3L
dependent and retain methylation above 25% in blastocyst.
Interestingly, although it has been suggested that intermediate density
or ‘weak’ CGIs would be most susceptible to regulation by
methylation (Borgel et al., 2010; Weber et al., 2007) we found that
loss of methylation at weak CGIs could not derepress most of the
genes examined: many of the loci identified by meDIP fall into this
category.
Significantly, there is good conservation in humans of the CGIs
that represent the gDMRs for both the testis and brain genes and
they are also heavily methylated in adult tissues, which is atypical
for most CGIs (Borgel et al., 2010). Conservation points to an
evolutionarily conserved requirement for methylation at these loci:
this is consistent with a regulatory role for the testis group, as they
can be derepressed following demethylation, as in mouse.
We also show that demethylation of genes in the postimplantation
group causes their transcriptional derepression in human fibroblasts,
which is the first demonstration, to the best of our knowledge, of this
in a normal human cell type. Likewise, we are not aware of any
previous report showing restoration of methylation at endogenous
single-copy sequences (as opposed to retroviral genes) by de novo
activity in an adult differentiated cell type. All of the genes
examined were more refractory to demethylation and activation in
the hTERT-1604 human fibroblasts than in the mouse NIH 3T3
fibroblasts, most likely because the latter, older cell line has become
more responsive to culture conditions over time. Greater
transcriptional response was seen to Aza, which inhibits all three
methyltransferases, than for DNMT1 siRNA alone, which might
reflect some dependence on DNMT3A/B for maintenance in these
cells as in mouse ESCs. Genes methylated postimplantation, such
as SYCP3, lost their methylation more readily and were reactivated
to a greater extent in the human cells than the testis gDMR
homologues, such as FKBP6, which might reflect underlying
differences in chromatin state between the two gene classes.
Although intragenic methylation has been reported previously for
some brain genes (Lister et al., 2013; Maunakea et al., 2010; Wu et
al., 2010), this is the first report, to our knowledge, to show that
neuronal genes represent the major class that inherits methylation
from the mother, with possible implications for transmission of
neuronal phenotypes. Notably, methylation at these loci is at
intragenic ‘orphan’ CGIs identified by CFP1 pulldown (Illingworth
et al., 2010; Smallwood et al., 2011). It has been suggested that
orphan CGIs can act as promoters early during development,
becoming inactivated and methylated later (Illingworth et al., 2010).
However, for the particular subset of orphan CGIs identified here as
carrying gametic marks, 82% show no evidence of being
transcriptional start sites in ESCs, this fraction rising to 89% in adult
brain (Fig. 1F). Consistent with this, their demethylation resulted in
no marked upregulation in steady-state mRNA levels. Recent reports
suggest that intragenic methylation in somatic tissues, particularly
at neuronal genes, might instead play a role in maintaining active
chromatin states (Wu et al., 2010) or in splice choice (Maunakea et
al., 2013). Preliminary analysis shows that this class of genes is
indeed significantly enriched for alternative splicing using the
DAVID ontology tool SP_PIR_KEY (46% of brain genes from the
RRBS dataset, P=2.4×10−13, FDR=3.0×10−10). Further investigation
of our brain gDMR set seems warranted given the conservation of
both positioning and methylation status for the promoter and
intragenic CGIs of the brain genes in human.
In addition to DNMT1 (Hirasawa et al., 2008; Howell et al.,
2001), ZFP57, a zinc finger-containing transcription factor, is also
vital for methylation maintenance at all imprinted loci and some
non-imprinted loci in preimplantation embryos and ESCs.
Interestingly, Fkbp6, which is a testis gDMR, was also bound by
ZFP57 and showed loss of methylation in KO cells (Quenneville et
al., 2011). When we analysed the sites bound by ZFP57 in genomewide ChIP studies we failed to uncover any other non-imprinted
gDMRs from our study, suggesting that other factors might be
involved in maintaining methylation at the other gDMR classes.
1321
Development
Fig. 7. Methylation dynamics and methyltransferase dependency of genes inheriting methylation from the mother. (Top) Methylation ontogeny of brain,
testis and imprinted gDMRs, as well as of postimplantation genes. The average methylation of each gene class (see key, bottom right) is shown: filled bar,
100% methylation; empty bar, 0% methylation. Methyltransferase requirements at different stages, where established, are shown within the central blue panel.
(Bottom) The effect of loss and restoration of methyltransferase activity in different experimental systems. The dish on the right represents ESCs.
RESEARCH ARTICLE
MATERIALS AND METHODS
Bioinformatic analysis and statistics
Published genome-wide methylation data from a number of studies (Borgel
et al., 2010; Kobayashi et al., 2012; Smallwood et al., 2011) were reexamined using publicly available tools. Ontology analysis including
statistical significance was performed in DAVID (Huang et al., 2009).
Transcription marks associated with orphan CGIs were derived from
Illingworth et al. (Illingworth et al., 2010). Comparison of gene features was
carried out in GALAXY (Giardine et al., 2005).
All laboratory experiments were carried out in triplicate, including at least
one biological repeat: graphs show a representative experiment, and error
bars represent s.e.m. Statistical analysis of results was carried out in Prism
(GraphPad) or Excel (Microsoft): pyrosequencing results were compared by
ANOVA, RT-qPCR results were analysed by t-test and bisulfite clonal
analysis results were compared by χ2 test.
Mouse strains
Derivation of Dnmt3l knockout oocytes/ovaries and matched WT tissues
was as previously described (Kaneda et al., 2004). All other mouse tissues
were derived from outbred TO mice (Harlan, Huntingdon, UK). Animal
work was carried out under licence from the appropriate regulatory bodies.
Cells
Culture media components are from Invitrogen unless otherwise stated. NIH
3T3 and hTERT-1604 fibroblasts were cultured in 4.5 g/l glucose D-MEM
supplemented with 10% FBS and 2× NEAA. Dnmt1 KO (± Dnmt1),
Dnmt3a/3b DKO ESCs and matched WT J1 cells were a kind gift of Dr M.
Okano (RIKEN Center for Developmental Biology, Kobe, Japan). ESCs
were maintained on Nunc plates (Davidson & Hardy, Belfast, UK) treated
with 0.1% gelatin (Sigma-Aldrich) and cultured in KnockOut D-MEM plus
15% KnockOut Serum Replacement, 1% ESC qualified FBS, 1× NEAA, 2
mM L-glutamine, 0.1 mM β-mercaptoethanol (Sigma-Aldrich) and 1000
U/ml LIF (Chemicon). For treatment with 5-aza-2′-deoxycytidine (Aza),
8×104 NIH 3T3 cells or 1×105 hTERT-1604 cells were seeded onto a 90 mm
plate in complete medium and allowed to attach overnight before replacing
with complete medium containing 1 μM Aza, which was renewed at 24-hour
intervals up to 72 hours.
siRNA treatment
siRNA treatments were carried out in 6-well plates, each well seeded with
1×106 hTERT-1604 cells or 1×105 NIH 3T3 cells, before reverse transfection
using Dharmafect 1 (Thermo Fisher Scientific) and 80 nM (NIH 3T3) or 100
nM (hTERT-1604) ON-TARGETplus SMARTpool DNMT1 siRNA or a
matched concentration of scrambled control (Thermo Fisher Scientific).
Cells were cultured in complete medium to allow recovery, with extraction
of RNA and DNA up to 28 days after addition of siRNA.
In situ hybridisation
In situ hybridisation was performed as previously described (Shovlin et al.,
2007). Primers used to generate probes are listed in supplementary material
Table S1.
Bisulfite treatment
Oocyte and sperm collection and isolation of DNA were as described (Li et
al., 2004; Walsh and Bestor, 1999). DNA was extracted from NIH 3T3 and
1322
hTERT-1604 cells using the Genomic DNA Purification Kit (Fermentas).
All other non-germ cell samples were incubated overnight at 55°C in lysis
buffer (50 mM Tris pH 8, 0.1 M EDTA, 0.5% SDS, 0.2 mg/ml proteinase
K) with rotation, and DNA was subsequently isolated by standard
phenol:chloroform extraction. Total DNA from 150-200 oocytes or 200-500
ng DNA from other samples was bisulfite converted using the EpiTect
Bisulfite Kit (Qiagen).
Methylation analysis
Bisulfite-treated DNA was PCR amplified in 1× buffer, 0.4 mM dNTPs, 1
μM primers (supplementary material Table S1), MgCl2 at a concentration
specific to the primer set and 0.01 U Taq DNA polymerase (reagents from
Invitrogen). Nested PCRs and COBRA were carried out as previously
described (Li et al., 2004) using BstUI (Dazl, Dpep3, Piwil1 and Snrpn) or
TaqαI (Csnka2ip, Fkbp6, H19, Rhox13 and Sycp3) enzymes from NEB.
Bisulfite sequencing of cloned PCR products in pCRII-TOPO vector
(Invitrogen) was performed using the ABI PRISM 3130 Genetic Analyzer
(Applied Biosystems). Pyrosequencing assays are from Qiagen
(supplementary material Table S2), except murine Adcy6, Crocc and Fkbp6
and human EFNA and GRIN3B, which were all designed in house using
PyroMark Assay Design software 2.0 (supplementary material Table S1).
Assays were conducted using the PyroMark PCR Kit (Qiagen); conditions
were: 95°C, 15 minutes; followed by 45 cycles of 94°C for 30 seconds,
56°C for 30 seconds and 72°C for 30 seconds; with final elongation at 72°C,
10 minutes. LUMA analysis was carried out as previously described (Karimi
et al., 2011a).
RNA extraction and RT-PCR
RNA from all samples was extracted using the RNeasy Mini Kit (Qiagen).
Reverse transcription reactions contained 300-500 ng total RNA, 0.5 μM
dNTPs, 0.5 μg oligo(dT) primer, 40 U RNaseOUT (Invitrogen), 1× reverse
transcriptase buffer (Fermentas) and 200 U RevertAid reverse transcriptase
(Fermentas) in a total volume of 20 μl. Reaction conditions were: 42°C, 50
minutes; 70°C, 15 minutes. cDNA was stored at −20°C until use.
Each RT-PCR contained 1× buffer, 0.4 mM dNTPs, 1 μM primers
(supplementary material Table S1), MgCl2 at a concentration specific to the
primer set and 0.01 U Taq DNA polymerase. The general PCR format was:
94°C, 3 minutes; followed by cycles of 94°C for 30 seconds, gene-specific
annealing temperature for 1 minute and 72°C for 1 minute; with final
elongation at 72°C, 5 minutes. RT-qPCRs were performed using 1×
LightCycler 480 SYBR Green I Master (Roche), 0.5 μM primers and 1 μl
cDNA. Reactions were run on the LightCycler 480 II (Roche), with an
initial incubation step of 95°C, 10 minutes; followed by 50 cycles of 95°C
for 10 seconds, 60°C for 10 seconds and 72°C for 10 seconds. Expression
was normalised to Hprt, and relative expression was determined using the
ΔΔCT method.
Acknowledgements
We are very grateful to Masaki Okano for the gift of cells and for insightful
comments. We thank Tanya Shovlin and Laszlo Hiripi for the in situ analysis; Hazel
Mangan for oocyte material; Heather McCook, Rhonda Black, Bernie O’Doherty
and Keith Thomas for technical support; and members of the C.P.W. group for
comments.
Competing interests
The authors declare no competing financial interests.
Author contributions
C.E.R. made initial observations, designed assays, assembled figures and
contributed to the manuscript; A.T. carried out the testis and some brain gDMR
work in mouse; R.E.I. analysed brain gDMR in mouse; K.M.O. analysed human
homologues and designed assays; S.S. and K.H. provided Dnmt3l−/− and WT
material; C.P.W. designed and supervised the study, carried out bioinformatic
analyses and wrote the manuscript.
Funding
Work in the C.P.W. laboratory was supported by grants from Invest Northern
Ireland [POC206] and the Medical Research Council [MR/J007773/1]. Deposited
in PMC for immediate release.
Development
Notably, Fkbp6 regained most methylation in 1KO+1 rescued ESCs,
which express ZFP57: at the same time, it was refractive to
remethylation in adult mouse and human fibroblasts, which are
predicted to lack this factor. At the testis gDMR loci generally,
recovery of methylation was coincident with re-establishment of
transcriptional repression. These results also have important
implications for the response of cells to demethylating agents.
In conclusion, we have identified two new classes of functionally
significant gDMRs that appear to be evolutionarily conserved and
might be important mediators of the phenotypic effects of pre- and
postnatal environmental exposures that alter DNA methylation levels.
Development (2014) doi:10.1242/dev.104646
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.104646/-/DC1
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